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gene expression Flashcards

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1
Q

upstream

A

before

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2
Q

downstream

A

after

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3
Q

RNA synthesis is catalyzed by

A

RNA pol using ssDNA as a template

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4
Q

core bacterial RNA polymerase

A

Core cannot directly interact with DNA - this function requires association of the core with a sigma factor
After initiation of transcription, sigma factor is released from RNAP holoenzyme

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5
Q

Strong promoters

A

Sigma factor makes sequence specific contacts with the promoter via the -10 and -35 regions
“Strength” of a promoter is dictated by affinity of sigma for these regions
Higher affinity = stronger promoter
Protein binding site is protected from DNAseI by protein binding

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6
Q

does RNA pol require a primer

A

RNA pol can catalyze de novo synthesis of polynucleotides (does NOT require a primer)
5’ end of the transcript will carry a triphosphate from the first NTP

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7
Q

RNAP uses ssDNA

A

RNAP maintains a region of ssDNA as it transcribes RNA

Transcription induces torsional stress, which is relieved by topoisomerases/gyrases

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8
Q

How are DNA replication and RNA transcription similar?

A

Both use ssDNA as template, template is always 3’ to 5’
Both make phosphodiester bonds RNApol uses NTPs DNApol uses dNTPs
Both synthesize nucleic acid 3’ to 5’

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9
Q

How are DNA replication and RNA transcription different?

A

RNApol starts at promoters, DNApol starts at origins of replication
But both have AT rich regions
DNA copies whole genome, RNA copies only discrete regions of genome
DNA ends with ds product, RNA ends with ss product

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10
Q

regulation of transcription in bacteria

A

Gene expression in bacteria is controlled by regulating whether or not a gene is expressed
Genes that work in common pathways are regulated together in units called operons

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11
Q

polycistronic

A

A single mRNA is made that codes for multiple proteins: polycistronic

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12
Q

operons

A

Operator and promoter are control regions (upstream of structural genes)

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13
Q

lacI

A

lacI = repressor protein

When lacI is bound to operator, transcription is off

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14
Q

lactose operon structural genes

A

lacZ, lacY, and lacA are structural genes: code for proteins needed to metabolize lactose

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15
Q

in vivo inducer

A

lactose is in vivo inducer

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16
Q

in vitro inducer

A

IPTG is lactose analog

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17
Q

no lactose

A

Without lactose, a repressor prevents transcription of the structural genes
lacI gene codes for lacI repressor protein
This protein is made constitutively (ALWAYS)

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18
Q

with lactose

A

With lactose, repressor binds lactose and releases the operator

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19
Q

binding site for lac repressor protein

A

Many DNA binding sites have palindromic symmetry

The region from -5 to +21 is protected from DNAseI when lacI is bound

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20
Q

what is required for full activation of lac operon

A

absence of glucose

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21
Q

absence of glucose

A

Glucose is a preferred carbon source and will be used completely before lactose
“Catabolite repression”
An increase in cAMP signals the absence of glucose
cAMP binds CAP, which binds the promoter and stimulates transcription

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22
Q

binding of CAP-cAMP induces,

A

DNA bending

Bending enhances RNAP holoenzyme binding

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23
Q

arabinose operon (araBAD

A

Arabinose is a pentose that can be a carbon source
Its degradation requires 3 enzymes found in the araBAD operon
Transcription is regulated by both catabolite repression and arabinose-based induction

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24
Q

araC gene

A

araC gene product binds arabinose and has a DNA binding domain
araC protein acts as BOTH an inducer and a repressor (depends on cellular conditions)

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25
araC with no arabinose
acts as a repressor The operon has 3 binding sites for araC, araO1, araO2, and araI (really 2 half sites) In absence of arabinose, araC protein forms a dimer that binds to araI1 and araO2 This keeps transcription of the operon off
26
araC with arabinose
araC is an inducer In presence of arabinose, araC releases the araO2 site and binds the araI2 site araC-arabinose interacts with CAP-cAMP (when glucose is absent) Together, this complex activates transcription
27
Tryptophan (trp) operon
Codes for 5 proteins needed to synthesize tryptophan Trp operon only transcribed when cellular trp levels are sufficiently low Regulated by trp repressor, which binds trp When trp repressor-trp complex is bound to the operator, the operon is off
28
trp operon attenuation
Operon is also subject to attenuation, which allows transcription to be regulated based on amount of available trp in the cell
29
in proks, how do translation and transcription occur
at the same time
30
leader sequence
This allows aa concentration to regulate operon’s level of transcription example of repressible negative regulation of gene expression assumes 2 diff secondary structures
31
2 structures of leader sequence of transcript
It trp levels are high, leader folds like a terminator, transcription STOPS (trp (the corepressor) binds trp repressor which blocks RNApol binding) If trp levels are low, the leader folds like an anti-terminator, transcription continues
32
attenuation: relatively high Trp levels
attenuation causing RNA pol to stop prematurely | Transcription = terminated
33
attenuation: relatively low Trp levels
The transcript folds like an anti-terminator | Transcription continues
34
Differences in transcription in eukaryotes
In euks, genomic DNA wraps around nucleosomes Access of RNA pol to DNA is more complex Euks have 3 RNA pols that transcribe different classes of genes RNAPI transcribes ribosomal RNAs, RNAP II transcribes protein-coding genes, RNAP III transcribes tRNA genes, some ribosomal RNA genes, and other small RNAs Regulation of transcription is more complicated in eukaryotes
35
RNA pol II (yeast)
C terminal domain (CTD) is important in regulation | Alpha-amanitin is an RNAP II-specific inhibitor
36
Promoters of eukaryotic protein-coding genes
TATA box (-25) is analogous to Pribnow box in proks Strong promoters have a CAAT box located about -80 Housekeeping genes have GC boxes
37
enhancers
Enhancers are positive regulators of gene transcription | They are bound by transcriptional activators
38
silencers
Silencers are DNA sequences that act as negative regulators They are bound by transcriptional repressors Location with respect to transcription start site varies Sequences are bidirectional - they function in either orientation
39
response elements
sequences found in promoters that are responsible to cellular conditions Response elements are bound by transcription factors that are activated by a particular cellular condition
40
basal levels of transcription requires
a complex of general transcription factors plus RNAP II
41
mediator
bridges transcription factors bound to enhances with the initiation complex Mediator interacts with RNAPII-CTD and can activate or repress transcription
42
DNA bending or looping
influences gene expression DNA looping allows additional proteins to interact at the initiation site and either stabilize RNAP binding or destabilize RNAP binding
43
access of transcription machinery to template strand
DNA is wrapped around nucleosomes (each nucleosome = 8 histone proteins) For access, must move nucleosomes (chromatin-remodeling complexes) and/or must reduce affinity between DNA and histones (histone-modifying enzymes)
44
chromatin-remodeling complexes
Contain proteins in SNF2 family of DEAD/H box-containing ATPases 3 complexes loosen DNA-nucleosome interactions by restructuring core octamers This gives RNAP II and general transcription factors access to promoters
45
histone-modifying enzymes
influence nucleosome-DNA affinity
46
histone acetyltransferases (HATs)
add acetyl groups to histone tails This separates DNA from nucleosomes (and promotes transcription) TFIID has HAT activity
47
methylation of histone tails
tightens DNA-nucleosome interactions (and represses transcription)
48
histone modifications
Post-translational modifications to histone tails can selectively recruit proteins to chromatin Proteins that cause compaction = repressed gene expression Proteins that cause relaxation = activates gene expression
49
important histone tail modifications
Lysine acetylation (HATs) and de-acetylation (HDACs) Lysine methylation and de-methylation (methyltransferases) Serine phosphorylation (kinases) Lysine ubiquitination Lysine sumolyation
50
model of transcriptional regulation
Gene activation first requires altering nucleosomes to relieve the repressive state of chromatin, followed by interactions of RNAPII/GTFs with promoters Transcriptional activators initiate the first step Mediator then facilitates the second step by bridging distant transcriptional activators and GTFs/RNAPII
51
DNA binding domains do what?
mediate interactions between proteins and DNA
52
DNA binding domains examples (3)
Helix-turn-helix (HTH) Zinc finger Leucine zipper-basic region (leucine zipper is a dimerization domain; basic region binds DNA)
53
helix-turn-helix motif
Alpha-helix fits into the major groove of B-form DNA One of the helices binds DNA It may recognize a specific sequence (direct readout) or a particular shape (indirect readout) HTH proteins act as dimers
54
Zn-finger motif
Zn is coordinated by Cys, His, or both A single protein may have many zinc fingers Each finger binds in the major groove of DNA
55
Leucine zipper
basic region proteins | NOT a DNA binding domain, but is often found in DNA binding proteins
56
capping of pre-mRNA
Guanylyl transferase catalyzes the addition of a guanylyl residue to the 5’ end of primary transcript (occurs co-transcriptionally)
57
methylation of the 5' end of pre-mRNA
This may facilitate later steps of pre-mRNA processing
58
Splicing requires precise removal of introns
Essential sequences include the exon/intron junction and the branch site (located within the intron)
59
snRNPs in spliceosomes
U1: targets 5’ splice U2: targets branch U4 to U6: targets 5’ splice, recruitment of branch point to 5’ splice site
60
Spliceosome assembly
snRNPs (protein and RNA) interact with pre-mRNA to form the spliceosome Splicing occurs co-transcriptionally
61
Alternative splicing
creates multiple protein isoforms from the same gene
62
DNA footprinting
methods to analyze protein–DNA complexes and to identify the position of the binding site

Biochemistry 2 (32 decks)

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